WO1991007982A1 - Treatment of hsv - Google Patents

Abstract

Basic fibroblast growth factor, a 146 amino acid residue polypeptide, analogs thereof, a lectin, suramin, protamine and/or a shortened fragment of either bFGF or of an analog thereof is used to prevent the spread of HSV-1. It has been found that topical application of an effective amount of such an agent prevents HSV uptake and/or infection in cells that are otherwise targets for such virus. One group of preferred agents are peptides which include the decapeptide sequence of a mammalian bFGF(106-115), e.g., bovine bFGF(106-115), examples of such preferred peptides include bovine bFGF(103-120)-NH2 and human bFGF(103-120)-NH2.

Description

TREATMENT OF HSV

This application is a continuation-in-part of our Application Serial No. 07/443,939, filed November 30, 1989.

The present invention relates generally to the prevention of the invasion of mammalian cells by viral nucleic acid, particularly to preventing the spreading of herpes simplex virus (HSV) and more particularly to the topical application of agents as a totally novel treatment for persons infected with HSV to suppress the uptake and infectivity of HSV.

Background of the Invention Herpes simplex virus type-1 (HSV-1) is a ubiquitous pathogen responsible for considerable morbidity in the general population. Infection by HSV-1 has become more and more of a problem throughout the United States and the world. Although a very substantial effort has been made to combat infection by HSV-1 and to prevent spread of the virus, no solution has yet been successful, and efforts are accordingly continuing.

The pathway for herpes simplex virus-l (HSV-1) entry into cells of epithelial, mesenchymal and neuronal origin remains undefined, as is the case with respect to other viral nucleic acid. Because HSV-1 is ubiquitous, causing morbidity for the general population and mortality for the immunocompromised host, the identification of those mechanisms that are responsible for its entry into cells is considered to be of paramount importance in order to develop strategies to prevent or alter the course of herpes virus-induced diseases. The putative sequence of events in herpes infection involves initial virion attachment to the cell surface through an interaction with cell-associated glycosaminoglycans (GAGs) , fusion of the viral envelope with the plasma membrane, de-envelopment and then release of the viral nucleocapsid into the cytoplasm of the cell. Although this viral attachment involves specific viral-associated glycoproteins and specific target cell binding sites and receptors, no specific receptor has yet been described for HSV-1.

Basic fibroblast growth factor (bFGF) is a potent itogen for both normal diploid fibroblasts and established cell lines, and studies confirm that, in addition to fibroblasts, bFGF is also mitogenic for a wide variety of normal diploid mesoderm-derived and neural crest-derived cells, including granulosa cells, adrenal cortical cells, chondrocytes, myoblasts, corneal and vascular endothelial cells from either bovine or human origin, vascular smooth muscle cells, and lens epithelial cells. bFGF has also been shown to substitute for platelet-derived growth factor in its ability to support the proliferation of fibroblasts exposed to plasma-supplemented medium. Consistent with its ability to stimulate the proliferation of bovine and human vascular endothelial cells, bFGF has a similar activity in vivo upon capillary endothelial cells and is considered an angiogenic factor. Fragments of bFGF have been discovered which are antagonistic to bFGF as reported by A. Baird et al., P.N.A.S.. 85. 2324 (1988) and by D. Schubert et al., J. Cell. Biol.. 104. 635-643 (1987) ; however, it was not heretofore expected that such fragments would have any anti-viral effect.

Summary of the Invention The present invention provides methods of preventing the infection of mammalian cells by viral nucleic acid and in particular preventing the spread of HSV by topical treatment with an agent that counteracts the invasive action of such virus. It is now believed that the bFGF receptor serves as a pathway for HSV entry and the entry of other viral nucleic acid into vertebrate cells. It has been found that bFGF, analogs of bFGF, and inhibitors of bFGF binding, including lectins, such as wheat germ agglutinin( GA) , suramin, protamine and certain competitive polypeptide antagonists of bFGF binding, prevent HSV-1 uptake and infectivity. bFGF is a 146 amino acid residue polypeptide having the sequence set forth hereinafter. The entire bFGF molecule, as well as the N-terminally extended versions thereof, may be employed in the present invention which utilizes topical application of such molecules or of certain fragments of basic fibroblast growth factor antagonistic to bFGF binding (hereinafter referred to as a group as bFGF peptides) to prevent the uptake and infectivity of HSV. In any case, the present invention utilizes the topical application of bFGF peptides and other agents that suppress the uptake and infectivity of HSV and other viral nucleic acid, presumably by occupying the bFGF receptors on target cells, particularly the high-affinity bFGF receptors. As used herein, topical should be understood to include -. application to the eye as well as application to the skin.

Pharmaceutical compositions useful in accordance with the teachings of the invention include such bFGF peptides and/or one of the other named agents, dispersed in a pharmaceutically acceptable liquid or solid carrier suitable for topical application. Such pharmaceutical compositions can be used in human clinical medicine, both to prevent the spread of virus to others and for application for therapeutic purposes for a person already infected with a virus such as HSV. If there are lesions to be healed, it may be preferable to apply the entire bFGF molecule or an agonist thereof that would assist in wound-healing; on the other hand, if at an early stage it was not felt desirable to close a wound, it may be preferable to use a bFGF antagonist or one of the other agents. These agents are considered effective to prevent the spread of other than HSV viruses that operate in a similar manner, e.g. HIV which is responsible for acquired immunodeficiency syndrome. Detailed Description of Certain Preferred Embodiments The invention preferably utilizes bFGF or certain fragments of mammalian bFGF, e.g. human or bovine basic FGF, or other agents, such as lectins, e.g. sura in and GA, to block the spread of HSV. The nomenclature used to define the bFGF peptides is that specified by Schroder & Lubke, "The Peptides", Academic Press (1965), wherein in accordance with conventional representation the residue having the free alpha-amino group at the N-terminus appears to left and the residue having the alpha-carboxyl group at the

C-terminus to the right. Where the amino acid residue has isomeric forms, it is the L-form of the amino acid that is represented. Human basic FGF is considered to be a 146-residue peptide having the following sequence (SEQ ID NO:l): Gly-Thr-Met-Ala-Ala-Gly-Ser-Ile-Thr-Thr-Leu

-11 -10 -5 -1

Pro-Ala-Leu-Pro-Glu-Asp-Gly-Gly-Ser-Gly-Ala-Phe-Pro-Pro-Gly-

5 10 15

His-Phe-Lys-Asp-Pro-Lys-Arg-Leu-Tyr-Cys-Lys-Asn-Gly-Gly-Phe-

20 25 30

Phe-Leu-Arg-Ile-His-Pro-Asp-Gly-Arg-Val-Asp-Gly-Val-Arg-Glu- 35 40 45

Lys-Ser-Asp-Pro-His-Ile-Lys-Leu-Gln-Leu-Gln-Ala-Glu-Glu-Arg-

50 55 60 Gly-Val-Val-Ser-Ile-Lys-Gly-Val-Cys-Ala-Asn-Arg-Tyr-Leu-Ala-

65 70 75

Met-Lys-Glu-Asp-Gly-Arg-Leu-Leu-Ala-Ser-Lys-Cys-Val-Thr-Asp-

80 85 90

Glu-Cys-Phe-Phe-Phe-Glu-Arg-Leu-Glu-Ser-Asn-Asn-Tyr-Asn-Thr-

95 100 105 Tyr-Arg-Ser-Arg-Lys-Tyr-Thr-Ser-Trp-Tyr-Val-Ala-Leu-Lys-Arg-

110 115 120

Thr-Gly-Gln-Tyr-Lys-Leu-Gly-Ser-Lys-Thr-Gly-Pro-Gly-Gln-Lys- 125 130 135

Ala-Ile-Leu-Phe-Leu-Pro-Met-Ser-Ala-Lys-Ser.

140 145 146

The C-terminus of the peptide is free acid. Bovine bFGF has the same formula except for the substitution of Ser for Thr in the 112-position and the substitution of Pro for Ser in the 128-position. The human molecule (as well as the bovine molecule) can have an N-terminal extension of up to 11 residues from the sequence without altering its effectiveness to combat HSV infection, e.g., the 9-residue extension: Met-Ala-Ala-Gly-Ser-Ile-Thr- Thr-Leu.

The present invention preferably utilizes a family of bFGF fragments which are each based upon a central core sequence from a native mammalian hormone bFGF. This core includes the ten residues appearing at positions 106-115 in the 146-residue molecule. In other words, relatively short peptides in the form of this ten residue sequence, as well as longer peptides, for example fragments of bFGF containing this decapeptide within their peptide sequence, show suppression of the uptake and infectivity of HSV when applied topically. Such bFGF fragments may be of very substantially increased lengths, relative to this decapeptide, as a result of the inclusion of N-terminal and/or C-terminal extensions of varying length—which should be evident from the fact that the entire bFGF molecule can be employed. The peptides are preferably, but not necessarily, a idated at the C-terminus. These extensions preferably, but not necessarily, comprise the particular residue sequences normally found at these locations in a native mammalian hormone; bFGF(116-146) is an example of a suitable C-terminal extension. Some substitutions may be made in the sequence at selected locations, as discussed hereinafter. More preferably, the extended fragment should include the sequence of bFGF(103-120) , a peptide which exhibits very good inhibition of HSV-1 uptake and infectivity.

The basis for the inhibition of HSV exhibited by these bFGF peptides is believed to be mediated by their binding interaction with the bFGF receptor, particularly the high-affinity bFGF receptor—see P.A. Wahche et al., J. Biol. Chem. 264, 4120 (1989) and T. Imamura et al., B.B.R.C. 155. 2, 583 (1988). Application of such bFGF peptide fragments, that show antagonism to mitogenesis jln vitro (including all bFGF target cell types) and that also prevent HSV-1 from binding to the bFGF receptor, may be preferred over using the entire bFGF molecule — which would also exhibit its own biopotency that might not be desirable in certain instances, e.g. where healing of lesions is not immediately desired. It now appears the minimum length peptide antagonist should contain the core sequence of bFGF(106-115) . However, in those instances where both anti-viral and wound-healing actions are desired, the employment of the entire bFGF molecule, or an equivalent N-terminally extended version thereof, or an equivalent analog thereof, such as acidic FGF, HST, FGF-5, FGF-6, INT/2 or KGF(FGF-7), all of which bind the FGF receptor, may be preferred.

To test the effectiveness of bFGF peptides to suppress the uptake and infectivity of HSV, variable concentrations of bFGF and fragments thereof were incubated with either human or bovine arterial smooth muscle cells for 2 hours; HSV-1 was then added. It was found that the treatment of these cells with recombinant bFGF inhibited the uptake of labeled (³H-thymidine;

20Ci/mmole) HSV-1 by the cells, measured as described in D. WuDunn et al., J. Virol. , 63, 52 (1989) which measures the penetration of HSV into the cell by measuring radioactivity, and it likewise decreased HSV infectivity as measured in the viral plaque assay described in R.A. Killington and K.L. Powell, Virology. B.W.J. Mahy (Ed), IRL Press, Washington, D.C., 1985, pp 207-236, which is a measure of whether the virus is replicating in the cells.

More specifically, bovine arterial smooth 1- muscle cells (sub-passage 3-5) , cultured as described in D.P. Hajjar et al., J. Clin. Invest. , 70, 469 (1982), were incubated for two hours at 4"C. with 50 nM of the peptide or protein being evaluated. These cell cultures were then inoculated with [³H-Tdr]-labeled virus(lμCi/10⁵ cells/well) at 37^βC. for three hours. The HSV-1 used was the type 1 strain F from facial vesicle and obtained from American Tissue Culture Association. To determine the uptake of virus, the cells were then washed twice with phosphate-buffered saline (PBS) , harvested on ice, and the amount of radioactivity that was cell-associated was determined by scintillation counting. Control incorporation (17,000 dpm/well) was used to normalize the effects of each additive, and triplicate determinations are made of each. To determine the infectivity of the virus, cell lysates were prepared 48 hours after the addition of HSV-1 to the cells and tested in the plaque assay. The results were normalized to the number of control plaques (33±6) . Each experiment was repeated at least twice, and the results represent the average values of triplicate cultures. In all instances, the standard deviations were less than 15%. Co-incubation of bFGF with HSV-1 at the time of infection produced substantially identical results, namely a decrease in uptake of about 67% of control and a decrease in infectivity of about 72%. In contrast, if bFGF was added 2 hours after viral inoculation, it failed to inhibit either virion uptake or infectivity. Additional experiments showed that these effects of bFGF were also highly specific and not a property of certain other native compounds present in the human body. Neither transforming growth factor-b(TFG-b) , nerve growth factor(NGF) , platelet-derived growth factor(PDGF) , colony stimulating factor(CSF) or glucagon, a polypeptide unrelated to cell growth, prevented viral uptake and/or infectivity. Other cells, including human umbilical vein endothelial cells, showed similar sensitivity to bFGF with respect to HSV-1 uptake and infectivity; however, bFGF had no effect on the uptake of adenovirus (AD-2) , an unrelated DNA virus. Collectively, these findings support the hypothesis that the inhibitory effects of bFGF on the uptake and infectivity of HSV-l are due to the occupancy of the high-affinity FGF receptor with concomitant blockage of a cellular portal of entry for HSV-1, and that the effects would be similar against any viral nucleic acid which uses the FGF receptor in its invasion of mammalian cells. The dose dependent effects of bFGF were also- examined with respect to the uptake and infectivity of HSV-1 virus. Three concentrations of bFGF were tested for their ability to inhibit uptake and infectivity of HSV-1. A concentration which was 100 nM did not significantly change the effect on uptake and infectivity from that which was measured using 50 nM; however, a decrease to 5 nM bFGF resulted in a suppression of uptake to only about 52% of control and of infectivity to only about 60%. Because similar inhibition of uptake was obtained at 4°C. and at 37°C, it can be concluded that the active peptides inhibit adsorption as well as penetration.

To distinguish whether HSV-1 competes with bFGF on the high or on the low affinity receptors, the following experiments were performed. Incubation of cells with a polypeptide fragment of bFGF that is known to interact with and occupy the high affinity FGF receptor, i.e. bFGF(103-120) , was found to inhibit HSV-1 uptake and infection. Finally, it is known that HSV-1 normally activates tyrosine kinase activity which is specifically associated with the high affinity bFGF receptor, and it is found that such activation is blocked by application of these agents. In contrast, peptide fragments that bind GAGs but not to the high affinity receptor, i.e. bFGF(l-24) and bFGF(121-146) , were found to have no effect on HSV-1 uptake. It is thus concluded that the high affinity receptor is the one of interest from the standpoint of HSV entry. Similar competitive binding assays with bFGF(103-120) can be used to determine whether a prospective agent, e.g. an analog of bFGF(1-146) , exhibits the capability of binding the high affinity FGF receptor.

A series of other compounds that had been found to inhibit bFGF binding to its receptor were also tested to determine their effect on suppressing the uptake and infectivity of HSV. Sura in, protamine and certain lectins, such as wheat germ agglutinin(WGA) or Triticum vulgaris were found to prevent HSV-1 uptake, and it is believed that other comparable lectins, exhibiting hemagglutinating activity, particularly those which bind to N-acetylglucosamine and polymers thereof, such as diacetylchitobiose and triacetylchitobiose, and those which are rich in cystine should have a similar effect. Histidine-rich glycoprotein(HRGP) and thrombospondin(TSP) fail to inhibit HSV-1 uptake. More specifically, bovine arterial smooth muscle cells (sub-passage 3-7) were incubated with histidine rich glycoprotein (HRGP, 50 ng/ml) , thrombospondin (TSP, 50 ng/ml) , anti-CR2 complement receptor (anti-C3d, 10 ng/ml) , dextran sulfate (DS, 50 μg/ml) , protamine (50 μg/ml) , Suramin (50 μg/ml) , wheat germ agglutinin (WGA, 50 μg/ml) and bFGF (103-120) (10 μg/ml) . Suramin was found to reduce the uptake of HSV-1 to about 23% of the control and the lower concentration of the bFGF peptide was found to reduce the uptake to about 30% of control. WGA was found to reduce the uptake of HSV-1 to about 62% and protamine to about 66% of control, whereas the remainder of the compounds tested were not felt to have a particularly significant effect on reducing HSV-1 uptake. In a further test using human arterial smooth muscle cells, protamine was found to reduce HSV-1 uptake to about 45% of control. The fact that two heparin-binding proteins, HRGP and TSP have no effect on HSV-1 uptake and infectivity supports the concept that the virion uses the high affinity receptor for bFGF rather than cell-associated GAGs.

Both uptake and infectivity were measured in further detail in cells treated with bovine bFGF(103-120) . In these instances, the bFGF peptides were added to cells either 2 hours prior to the addition of HSV-1 or simultaneously with the addition of virus. Control incorporation was used to normalize the effects of each additive, and triplicate determinations were made at each dosage. Each experiment was repeated twice, and the standard deviations were less than 10%. There is some slight improvement, generally between about 10% and about 20%, in inhibition of uptake and of infectivity at relatively low concentrations, between about 1 μM and about 5 μM, when the bFGF is added prior to rather than simultaneously with the addition of HSV-1.

It has also been found that HSV-1 can displace the binding of radiolabelled bFGF to its high affinity receptor. When cross-linking experiments were performed in the presence of HSV-1, bFGF could no longer be used to identify the receptor. It has further been shown that HSV-1 and bFGF are only competing for the high affinity receptor. The present results establish that HSV-1 activates the high affinity FGF receptor and uses it to enter target cells. This transmembrane protein which constitutes the high affinity receptor has been extensively described, and a full length cDNA clone has recently been isolated and sequenced, Lee, P.L., Science, 245. 57 (1987) . Although it is difficult to establish whether the FGF receptor is the only portal of entry, it is clearly responsible for as much as 75% of viral uptake into the cells examined thus far. These results are considered to be consistent with the findings of others who have attributed 20-30% of viral penetration as being "non-specific". There are at least two molecular weight forms of the FGF receptor, and HSV-1 appears to affect both. Thus, it seems likely that either form can serve as a portal of cellular entry. The FGF receptor is present on the cell surface as an integral membrane protein of many cells in culture, a finding that is consistent with the in vitro tropism of HSV-1. The distribution and expression of the FGF receptor in vivo is not well known and thus cannot, at this time, correlate with the known in vivo tissue-tropism of HSV-1. However, one of the richest sources of FGF receptor is the nervous system, which is a well described locus of HSV-1 infection and latency.

Following the identification of a high-affinity basic FGF receptor and isolation and sequencing of a full-length cDNA clone, it was determined that CHO cells contain fewer than 1000 FGF receptors per cell (background levels) . Such cells do not respond mitogenically to bFGF and are unable to take up HSV-1. CHO cells were transfected with the FGF receptor gene to create targets for HSV-1 uptake; transfection was carried out using an expression vector containing the cDNA of the mouse homolog of the chicken basic FGF receptor cDNA, and two stable transfectant lines were isolated, which were found to express 31,000 and 100,000 high-affinity FGF receptors per cell, respectively. There was a greater than tenfold increase in the uptake of [³H]HSV-1 by the transfected cells, compared to the parental cell line. The FGF peptide FGF(103-120) that binds to the high- affinity FGF receptor, blocked this increase. Despite increased uptake of HSV-1, the transfected cells did not develop cytopathologic effects, indicating that there may be another block to viral replication. Furthermore, binding experiments at 4*C showed that 60% of HSV binding to these FGF receptor-expressing cells was sensitive to displacement by prolonged (30-minute) washing with dextran sulfate (1 mg/ml) . The residual bound radioactivity was increased by greater than tenfold (disintegrations per minute) in these FGF receptor- expressing cell lines, relative to controls, indicating that the virus was bound to the receptor. These data also suggest that heparan sulfate may help to stabilize the binding of virions to the receptor. By contrast, binding studies with the FGF receptor-expressing cell lines showed no differences in low-affinity (heparan sulfate) binding of ¹²⁵I-labeled basic FGF relative to the wild-type cell lines. These findings further support the conclusion that HSV-1 is bound and internalized by the high-affinity receptor.

The concept that HSV-1 utilizes the basic FGF receptor is an extension of the recent demonstration that HSV-1 binds heparan sulfate on the cell surface as the "initial" interaction for viral adsorption to the cell surface. This mechanism is thus almost identical to that proposed for basic FGF interactions with target cells. In the case of HSV-1, the loss by cells of infectivity after heparinase treatment most likely reflects the decreased efficiency of virion delivery and binding to the basic FGF high-affinity receptor.

The mechanism by which the HSV-1 virion recognizes the basic FGF receptor is undefined; however experiments reported hereinafter provide the basis for a conclusion in this respect. HSV may bind basic FGF on its surface during assembly of the virion or may bind FGF released into the extracellular environment during normal cell turnover and thereby use FGF as a vehicle for binding to the receptor. In support of this concept, density gradient-purified HSV-1 tests strongly positive for basic FGF in a protein immunoblot. As has been proposed for human cyto egalovirus, this would be a mechanism where a virus interacts with a natural ligand to facilitate receptor-mediated uptake. This mechanism would also explain why heparan sulfate on the cell surface facilitates the adsorption of HSV, because FGF is a strong heparin-binding protein. Alternatively, a direct interaction between the HSV envelope glycoprotein and the FGF receptor may also contribute to the ability of HSV to penetrate cells via the FGF receptor.

Incubation of Swiss 3T3 fibroblasts with HSV-1 results in the tyrosine phosphorylation of a unique 90-kD protein substrate that characterizes the cellular response to basic FGF; the DNA virus, adenovirus, has no effect. Because an immunoneutralizing antibody to bFGF inhibits the HSV-1 dependent phosphorylation of the 90-kD protein, it appears that HSV-1 either releases bFGF from target cells or that a bFGF-like protein is associated with the viral particle.

There are no known proteins encoded in the HSV- 1 genome that have significant (>25%) structural homology with fragments of bFGF; thus, it is difficult to account for the virion's capacity to bind and activate the FGF receptor. However, immunoneutralizing antibodies to bFGF can prevent viral uptake into target cells, and a bFGF- like epitope may exist on the virion surface. Several viruses have been shown to use the binding sites for known physiological ligands as portals of entry into target cells, and it is not always known how they recognize these receptors. HSV-1 appears to be distinct from the only other known virus that uses a growth factor receptor to penetrate target cells; the vaccinia stomatitis virus (VSV) encodes a protein which has homology with epidermal growth factor(EGF) which it uses to bind to the EGF receptor of cells. The rhinovirus utilizes the receptor for the cell adhesion molecule ICAM-1 to penetrate cells, and the HIV virus utilizes the CD4 glycoprotein receptor. The rabies virus infects cells through the acetylcholine receptor, and the reovirus enters through the j3-adrenergic receptor. The portal of entry of HSV-1 is also distinct from the other members of the herpes viridae family in that Epstein-Barr virus recognizes the C3d complement receptor and HSV-1 does not. Thus, different members of the herpes family appear to be targeting cells by different portals of entry.

Western blot analyses (N=4) of density-gradient purified virus has established the presence of an 18-kD immunoreactive protein that is indistinguishable from human recombinant bFGF. Because layers of mock infectέd cells or of purified adenovirus are devoid of any similar protein, this may explain the ability of HSV-1 to recognize the bFGF receptor by having bFGF (of presumably cellular origin) associated with the virion. Although, as indicated, the mechanism through which a host-cell- derived bFGF becomes associated with the replicating viral particle is not known, the virus most likely associates with the growth factor at the time of virion budding from the nuclear membrane because this is one of the locations where the 18-kD form of bFGF has been localized. Prior to viral shedding, an association may develop between the host-cell-derived bFGF and the newly synthesized HSV-1 virion that ultimately gives the viral particle the capacity to interact with the bFGF receptor on other target cells. Thus, HSV-1 is like VSV in being able to incorporate host cell surface proteins into the virion. Thus, HSV-1, like cytomegalovirus, may use these target cell-derived proteins to confer infectivity. Inasmuch as herpes virus latency is promoted by nerve growth factor in vitro, it is particularly interesting that the same virus uses bFGF, another brain-derived neurotrophic factor, for cell penetration.

The identification of the FGF receptor as the site of entry of HSV-1 into target cells establishes a critical mechanism to explain how HSV-1 enters the cell, and the availability of specific receptor antagonists that can prevent HSV-1 uptake provides promise in controlling the spread of HSV-1 infection and its related pathology. They provide similar promise in combatting other viral nucleic acids which use the FGF receptor as a portal of entry into mammalian cells.

One family of bFGF peptide fragments and analogs thereof useful in the method of the invention is conveniently expressed by the following formula, which is based on the naturally occurring sequence of mammalian bFGF(93-146) , e.g. human, bovine, rat, etc. (SEQ ID NO:2) :

Phe-Phe-Phe-Glu-Arg-Leu-Glu-Ser-Asn-Asn-Tyr-Asn-Thr- Tyr-Arg-Ser-Arg-Lys-Tyr-Xaa-Xaa-Xaa-Xaa-Val-Ala-Leu-Lys- Arg-Thr-Gly-Gln-Tyr-Lys-Leu-Gly-Xaa-Lys-Thr-Gly-Pro-Gly- Gln-Lys-Ala-Ile-Leu-Phe-Leu-Pro-Met-Ser-Ala-Lys-Ser, wherein Xaa in the 20-position is Ser, Thr or D-Ser, Xaa in the 21-position is Ser, Ala or D-Ser, Xaa in the 22- position is Trp or Met, Xaa in the 23-position is Tyr or Phe, and Xaa in the 36-position is Pro or Ser and wherein one or more of the residues in positions 14 through 19, inclusive, can be substituted by its D-isomer; provided however that from 1 to 13 residues, in sequence, can be deleted beginning at the N-terminus, that from 1 to 30 residues, in sequence, can be deleted beginning at the C-terminus; that an extension of up to 91 residues, based upon a native mammalian bFGF peptide, can be included at the N-terminus; and that the C-terminus may be amidated. Because the entire molecule can also be used, it should be understood that extensions to the N-terminus of the above formula can be included to create effective peptide versions which are shorter than the native 146-residue molecule or even slightly longer (i.e., by up to 11 residues) . These peptides bind to the FGF receptor and are effective in suppressing the uptake and infectivity of HSV infection. As mentioned hereinbefore, peptides as short as ten residues in length which include the core sequence bFGF(106-115) are effective to inhibit the uptake and infectivity of HSV-1. If residues are added to either or both termini of this decapeptide, the peptide remains effective but may result in some receptor activation and agonist activity, and if such is not desired, peptides shorter than the native molecule may be preferred.

It may be preferable to synthesize peptides which include only naturally occurring residues and which are about 45 amino acids or greater in length by using- recombinant DNA methods. On the other hand, it may be preferable to synthesize peptides of about 30 residues or less in length using the well-known chain elongation techniques, such as solid-phase synthesis, as on a Merrifield resin or the like.

To synthesize a bFGF peptide containing only naturally occurring amino acid residues by recombinant DNA, a double-stranded DNA chain which encodes the desired amino acid sequence is synthetically constructed, as is well known in the art. The degeneracy of the genetic code permits a wide variety of codon combinations to be used to form the DNA chain that encodes the product polypeptide. Certain particular codons are more efficient for polypeptide expression in certain types of organisms, and the selection of codons preferably is made according to those codons which are most efficient for expression in the type of organism which is to serve as the host for the recombinant vector. However, any correct set of codons should encode the desired product, even if slightly less efficiently. Codon selection may also depend upon vector construction considerations; for example, it may be necessary to avoid creating a particular restriction site in the DNA chain if, subsequent to insertion of the synthetic DNA chain, the vector is to be manipulated using a restriction enzyme that cleaves at such a site. Also, it is necessary to avoid placing restriction sites in the DNA chain if the host organism which is to be transformed with the recombinant vector containing the DNA chain is known to produce a restriction enzyme that would cleave at such a site within the DNA chain.

In addition to the bFGF peptide-encoding sequences, the DNA chain that is synthesized may contain additional sequences, depending upon vector construction considerations. Typically, a DNA chain is synthesized with linkers at its ends to facilitate insertion into - restriction sites within a cloning vector. The DNA chain may be constructed so as to encode the desired sequence as a portion of a fusion polypeptide; and if so, it will generally contain terminal sequences that encode amino acid residue sequences that serve as proteolytic processing sites, whereby the desired polypeptide may be proteolytically cleaved from the remainder of the fusion polypeptide. The terminal portions of the synthetic DNA chain may also contain appropriate start and stop signals.

To assemble the desired DNA chain, oligonucleotides are constructed by conventional methods, such as procedures described in T. Maniatis et al., Cold Spring Harbor Laboratory Manual, Cold Spring Harbor, New York (1982) (hereinafter, CSH) . Sense and antisense oligonucleotide chains, up to about 70 nucleotide residues long, are synthesized, preferably on automated synthesizers, such as the Applied Biosystem Inc. model 380A DNA synthesizer. The oligonucleotide chains are constructed so that portions of the sense and antisense oligonucleotides overlap, associating with each other through hydrogen bonding between complementary base pairs and thereby forming double stranded chains, in most cases with gaps in the strands. Subsequently, the gaps in the strands are filled in and oligonucleotides of each strand are joined end to end with nucleotide triphosphates in the presence of appropriate DNA polymerases and/or with ligases.

The double-stranded bFGF peptide-encoding DNA chain is then modified as necessary to permit its insertion into a particular appropriate cloning vector in mind. The cloning vector that is to be recombined to incorporate the DNA chain is selected appropriate to its viability and expression in a host organism or cell line, and the manner of insertion of the DNA chain depends upon factors particular to the host — all as well known in^" the art and set forth in detail in U.S. application Serial No. 270,225, filed November 14, 1988 in the name of A. Baird et al., the disclosure of which is incorporated herein by reference. Most peptides can be synthesized by suitable chain elongation or coupling-type methods, such as by exclusively solid-phase techniques, by partial solid-phase techniques, by fragment condensation or by classical solution couplings. Common to coupling-type syntheses is the protection of the labile side-chain groups of the various amino acid moieties with suitable protecting groups which will prevent a chemical reaction from occurring at that site until the group is ultimately removed. Usually also common is the protection of an alpha-a ino group on an amino acid or a fragment while that entity reacts at the carboxyl group, followed by the selective removal of the alpha-amino protecting group to allow subsequent reaction to take place at that location. Details of such chain elongation processes are set forth in the aforementioned U.S. patent application. The following Example 1 sets forth a preferred method for synthesizing a bFGF fragment by the solid-phase technique. It will of course be appreciated that the synthesis of a correspondingly shorter peptide fragment is effected in the same manner by merely eliminating the requisite number of amino acids at either end of the chain and a longer peptide by appropriately adding residues.

EXAMPLE 1 The synthesis of bovine bFGF(103-l20) -NH₂ is conducted in a stepwise manner using a Beckman 990 Peptide Synthesizer and an MBHA resin. Coupling of BOC-Arg(Tos) to the resin is performed by the general procedure set forth in U.S. Patent No. 4,292,313, and the synthesis proceeds as set forth in detail in the aforementioned U.S. patent application.

Following cleavage and deprotection, the peptide is subjected to gel filtration, cation-exchange chromatography and preparative HPLC on a Vydec C₄ column using a 0.1% TFA and acetonitrile solvent system. Purification details are generally set forth in Ling et al. Biochem. Biophvs. Res. Commun. 95, 945 (1980) . Testing of the purified peptide shows that, as set forth hereinbefore, the peptide inhibits HSV-1 uptake of and infectio in bovine arterial smooth muscle. The synthesis is repeated using a chloromethylated resin to produce the same peptide having a free acid C-terminus, generally following the procedure described in Biopolymers. 12, 2513-19 (1973) to link Arg to the chloromethylated resin. Testing in the manner described hereinbefore shows that the peptide having the free acid N-terminus inhibits HSV-1 uptake of and infection in bovine arterial smooth muscle.

EXAMPLE 2 The synthesis of bovine bFGF(93-120)-NH₂ is conducted in a stepwise manner using a Beckman 990 synthesizer and an MBHA resin in the manner described in Example 1. Testing in the manner described hereinbefore shows that the peptide inhibits HSV-1 uptake of and infection in bovine arterial smooth muscle. EXAMPLE 3

The synthesis of bovine bFGF(106-118)-NH₂ is conducted in a stepwise manner using a Beckman 990 synthesizer and an MBHA resin in the manner described in Example 1. Testing in the manner described hereinbefore shows that the peptide inhibits HSV-1 uptake of and infection in bovine arterial smooth muscle.

EXAMPLE 4 The synthesis of bovine bFGF(103-146)-NH₂ is conducted in a stepwise manner using a Beckman 990 synthesizer and an MBHA resin in the manner described in Example 1. Testing in the manner described hereinbefore shows that the peptide inhibits HSV-1 uptake of and infection in bovine arterial smooth muscle.

EXAMPLE 5 The synthesis of bovine bFGF(97-120)-NH₂ is conducted in a stepwise manner using a Beckman 990 synthesizer and an MBHA resin in the manner described in Example 1. Testing in the manner described hereinbefore shows that the peptide inhibits HSV-1 uptake of and infection in bovine arterial smooth muscle.

EXAMPLE 6 The synthesis of bovine bFGF(100-120)-NH₂ is conducted in a stepwise manner using a Beckman 990 synthesizer and an MBHA resin in the manner described in Example 1. Testing in the manner described hereinbefore shows that the peptide inhibits HSV-1 uptake of and infection in bovine arterial smooth muscle.

EXAMPLE 7 The synthesis of bovine bFGF(106-120)-NH₂ is conducted in a stepwise manner using a Beckman 990 synthesizer and an MBHA resin in the manner described in Example 1. Testing in the manner described hereinbefore shows that the peptide inhibits HSV-1 uptake of and infection in bovine arterial smooth muscle. EXAMPLE 8

The synthesis of [Met¹¹⁴]-bovine bFGF(106-120)-NH₂ is conducted in a stepwise manner using a Beckman 990 synthesizer and an MBHA resin in the manner described in Example 1. Testing in the manner described hereinbefore shows that the peptide inhibits HSV-l uptake of and infection in bovine arterial smooth muscle.

EXAMPLE 9 The synthesis of [Phe¹¹⁵]-bovine bFGF(106-120)-NH₂ is conducted in a stepwise manner using a Beckman 990 synthesizer and an MBHA resin in the manner described in Example 1. Testing in the manner described hereinbefore shows that the peptide inhibits HSV-l uptake of and infection in bovine arterial smooth muscle.

EXAMPLE 10 The synthesis of bovine bFGF(106-115)-NH₂ is conducted in a stepwise manner using a Beckman 990 synthesizer and an MBHA resin in the manner described in Example 1. Testing in the manner described hereinbefore shows that the peptide inhibits HSV-1 uptake of and infection in bovine arterial smooth muscle.

EXAMPLE 11 The synthesis of [Ala¹¹³]-bovine bFGF(103-146)-NH₂ is conducted in a stepwise manner using a Beckman 990 synthesizer and an MBHA resin in the manner described in Example I. Testing in the manner described hereinbefore shows that the peptide inhibits HSV-1 uptake of and infection in bovine arterial smooth muscle.

EXAMPLE 12 The syntheses of the following compounds, based upon the bovine sequence, are conducted in a stepwise manner using a Beckman 990 synthesizer and an MBHA resin in the manner described in Example 1: bFGF(106-125)-NH₂, " (106-130)- " , " (106-135)- " ,

" (106-140)- " , " (106-146)- " . The above five syntheses are once repeated to create these same five fragments based on the human sequence. The syntheses are then again repeated using a chloromethylated resin to create these same five bovine fragments having free acid at the C-termini, namely: bFGF(106-125)-OH, " (106-130)- " " (106-135)- "

" (106-140)- " " (106-146)- "

Testing in the manner described hereinbefore shows that each of the fifteen peptides inhibits HSV-1 uptake of and infection in bovine arterial smooth muscle.

EXAMPLE 13 The syntheses of the following compounds are conducted in a stepwise manner using a Beckman 990 synthesizer and an MBHA resin in the manner described in Example 1: [ D-Tyr 106 -bFGF ( 106-120) -NH₂ I

[ D-Arg 107 II [D-Ser 108 [D-Arg 109 [ D-Lyε 110 [ D-Tyr 111 [D-Ser 112 II and [ D-Ser 113 II

Testing in the manner described hereinbefore shows that each peptide inhibits HSV-1 uptake of and infection in bovine arterial smooth muscle.

EXAMPLE 14 Bovine acidic FGF (aFGF) is 140 amino acid residues in length and is extracted and purified by the protocol described in F. Esch et al., B.B.R.C.. 133. 554 (1985) , wherein the entire amino acid sequence is set forth. Testing of aFGF in the manner described hereinbefore shows that the peptide inhibits uptake of HSV-1 and HSV-1 infection in bovine arterial smooth muscle.

EXAMPLE 15 Bovine acidic FGF is also found in a form lacking the N-terminal 6 residues, aFGF (7-140) , as described in G. Gimenez-Gallego et al., Science, 230, 1385 (1985) . Testing of aFGF (7-140) in the manner described hereinbefore shows that the peptide inhibits uptake of HSV-1 and HSV-1 infection in bovine arterial smooth muscle.

EXAMPLE 16 Human acidic FGF is highly homologous to bovine aFGF and can be extracted and purified using the same protocol, see G. Gimenez-Gallego et al., B.B.R.C.. 135, 541 (1986) . Testing of human aFGF in the manner described hereinbefore shows that the peptide inhibits uptake of HSV-1 and HSV-1 infection in bovine arterial smooth muscle. Because it appears that the growth of human melanomas and other melanocytes is promoted by bFGF, in some instances, it may be preferred to employ a bFGF antagonist peptide or a lectin or a protamine or suramin, instead of the complete or substantially complete bFGF molecule. Such is also the case when it is desired to inhibit repair of lesions. Synthetic bFGF peptides and/or a suitable lectin, and/or a protamine and/or suramin combined with a pharmaceutically acceptable carrier to form a pharmaceutical composition, are preferably topically administered to mammals, including humans. The dosage or concentration employed may vary with the particular condition being treated, with the severity of the condition and with the duration of desired treatment.

These agents, which are capable of binding with the FGF receptors on mammalian cells in a manner to block the site on such receptor used by certain viral nucleic acid for attachment and entry, will likely be administered dispersed in an appropriate pharmaceutically acceptable carrier suitable for topical application. Such pharmaceutical compositions can be used to prevent the spread of such viruses, particularly HSV-I, to other mammals not infected with the virus or to prevent its spread to yet unaffected areas of a mammal locally infected with such a virus. For human use, it may be preferred to combine the agent with a lipstick-type or chapstick-type base that is designed to be applied by rubbing. Peptides of this general type can be administered in the form of pharmaceutically acceptable nontoxic salts, such as acid addition salts or metal complexes, e.g., with zinc or iron, which forms are considered to be equivalents. Illustrative acid addition salts include hydrochloride, hydrobromide, sulphate, phosphate, maleate, acetate, citrate, benzoate, succinate, malate, ascorbate, tartrate and the like.

Although the invention has been described with regard to its preferred embodiments, which constitute the best mode presently known to the inventors, it should be understood that various changes and modifications as would be obvious to one having the ordinary skill in this art may be made without departing from the scope of the invention which is set forth in the claims appended hereto. Any mammalian bFGF or an appropriate receptor-binding antagonist thereof can be employed; for example, either Thr or Ser can be present in what would be position 112 of such a bFGF peptide, based upon the 146-residue native peptide. Moreover, either Asp or Glu can be in position 90, and either Ser or Pro can be in position 128. As also indicated hereinbefore. Met can be substituted for Trp in the 114-position, Phe can be substituted for Tyr in the 115-position, and Ala can be substituted for Ser in the 113-position. Other similar substitutions as would be obvious to one skilled in peptide chemistry may be present that provide equivalent FGF antagonists or FGF agonists. As mentioned hereinbefore, equivalent analogs of mammalian bFGF(1-146) can be employed, including the N-terminally extended molecules, acidic FGF, HST, INT/2, FGF-5, FGF-6 and KGF(FGF-7) so long as they bind the FGF receptor. Generally, the residue Tyr can be added at either terminus of a synthetic bFGF peptide without substantially affecting the receptor-binding effect of that particular peptide, which is considered to be of primary importance for purposes of the present invention. Inasmuch as the function of the bFGF peptide, when it is employed, is primarily one of binding, it is the sequence that is most important, and the C-terminus can be free acid, amide or an equivalent moiety. Specific features of the invention are emphasized in the claims which follow.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: Baird, J. A.

Hajjar, David P.

(ii) TITLE OF INVENTION: Treatment of HSV

(iii) NUMBER OF SEQUENCES: 2

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Fitch, Even, Tabin & Flannery

(B) STREET: 135 South LaSalle Street, Suite 900

(C) CITY: Chicago

(D) STATE: Illinois

(E) COUNTRY: USA

(F) ZIP: 60603

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentln Release #1.24

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER:

(B) FILING DATE:

(C) CLASSIFICATION:

(vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: US 07/443,939

(B) FILING DATE: 30-NOV-1989

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Schumann, James J.

(B) REGISTRATION NUMBER: 20856

(C) REFERENCE/DOCKET NUMBER: 50742

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: (619)552-1311

(B) TELEFAX: (619)552-0095

(2) INFORMATION FOR SEQ ID NO:l:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 157 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:

Gly Thr Met Ala Ala Gly Ser lie Thr Thr Leu Pro Ala Leu Pro Glu 1 5 10 15

Asp Gly Gly Ser Gly Ala Phe Pro Pro Gly His Phe Lys Asp Pro Lys 20 25 30

Arg Leu Tyr Cys Lys Asn Gly Gly Phe Phe Leu Arg lie His Pro Asp 35 40 45

Gly Arg Val Asp Gly Val Arg Glu Lys Ser Asp Pro His lie Lys Leu 50 55 60

Gin Leu Gin Ala Glu Glu Arg Gly Val Val Ser He Lys Gly Val Cys 65 70 75 80

Ala Asn Arg Tyr Leu Ala Met Lys Glu Asp Gly Arg Leu Leu Ala Ser 85 90 95

Lys Cys Val Thr Asp Glu Cys Phe Phe Phe Glu Arg Leu Glu Ser Asn 100 105 110

Asn Tyr Asn Thr Tyr Arg Ser Arg Lys Tyr Thr Ser Trp Tyr Val Ala 115 120 125

Leu Lys Arg Thr Gly Gin Tyr Lys Leu Gly Ser Lys Thr Gly Pro Gly 130 135 140

Gin Lys Ala He Leu Phe Leu Pro Met Ser Ala Lys Ser 145 150 155

(2) INFORMATION FOR SEQ ID NO:2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 54 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:

Phe Phe Phe Glu Arg Leu Glu Ser Asn Asn Tyr Asn Thr Tyr Arg Ser 1 5 10 15

Arg Lys Tyr Xaa Xaa Xaa Xaa Val Ala Leu Lys Arg Thr Gly Gin Tyr 20 25 30

Lys Leu Gly Xaa Lys Thr Gly Pro Gly Gin Lys Ala He Leu Phe Leu 35 40 45

Pro Met Ser Ala Lys Ser 50

Claims

WHAT IS CLAIMED IS:

1. A method of preventing the uptake and infectivity of HSV in mammals, which method comprises administering an effective amount of one or more agents having the capability of binding the high affinity receptor of FGF.

2. A method according to Claim 1 wherein said agent is selected from the group consisting of bFGF, analogs of bFGF, lectins, protamines, suramin and peptides having the following formula SEQ ID NO:2: Phe-Phe-Phe-Glu-Arg-Leu-Glu-Ser-Asn-Asn-Tyr-Asn-Thr- Tyr-Arg-Ser-Arg-Lys-Tyr-Xaa-Xaa-Xaa-Xaa-Val-Ala-Leu-Lys- Arg-Thr-Gly-Gln-Tyr-Lys-Leu-Gly-Xaa-Lys-Thr-Gly-Pro-Gly- Gln-Lys-Ala-Ile-Leu-Phe-Leu-Pro-Met-Ser-Ala-Lys-Ser, wherein Xaa in the 20-position is Ser, Thr or D-Ser, Xaa in the 21-position is Ser, Ala or D-Ser, Xaa in the 22- position is Trp or Met, Xaa in the 23-position is Tyr or Phe, Xaa in the 36-position is Pro or Ser, and one or more of the residues in positions 14 through 19 can be substituted by its D-isomer; provided however that the N- terminus can be shortened by deleting from 1 to 13 residues in sequence, that the C-terminus can be shortened by deleting from 1 to 30 residues in sequence, that an extension of up to 91 residues in the form appearing in a native mammalian bFGF peptide can be added at the N-terminus, and that the C-terminus can be optionally amidated.

3. A method according to Claim 2 wherein said agent is a peptide which has the sequence of either bovine bFGF (93-120) or human bFGF (93-120) .

4. A method according to Claim 2 wherein said agent is a peptide which has the sequence of bovine bFGF (106-115) or human bFGF (106-115) .

5. A method according to Claim 2 wherein said agent is a peptide which has the sequence of bovine bFGF

(103-120) or human bFGF (103-120) .

6. A method according to Claim 1 wherein said agent is a mammalian bFGF.

7. A method according to Claim 1 wherein said agent is human bFGF.

8. A method according to Claim 1 wherein said agent is human bFGF (1-146) having at its N-terminus an extension of Met-Ala-Ala-Gly-Ser-Ile-Thr-Thr-Leu, or an N-terminally shortened version thereof.

9. A method according to Claim 1 wherein said agent is WGA.

10. A method according to Claim 1 wherein said agent is suramin.

11. A method according to Claim 1 wherein said agent is a protamine.

12. A method of treatment according to Claim 1 wherein said administering is by topical application.

13. A method according to Claim 12 wherein said administering is to a mammal already infected with HSV.

14. A method according to Claim 12 wherein said administering is to a mammal not infected with HSV.

15. A method for preventing the attachment of viral nucleic acid to, and subsequent release of viral nucleic acid into, mammalian cells not containing said viral nucleic acid, which method comprises administering an effective amount of an agent capable of binding with the FGF receptor sites on said cells so as to block attachment of said viral nucleic acid thereto.

16. A method according to Claim 15 wherein said agent is a peptide which includes the sequence of bovine bFGF (106-115) or human bFGF (106-115) .

17. A method according to Claim 15 wherein said agent is a mammalian bFGF.

18. A method according to Claim 15 wherein said agent is human bFGF.

19. A method according to Claim 15 wherein said agent is WGA, suramin or a protamine.

20. An agent selected from the group consisting of bFGF, analogs of bFGF, lectins, protamines, suramin and peptides having the following formula SEQ ID NO:2: Phe-Phe-Phe-Glu-Arg-Leu-Glu-Ser-Asn-Asn-Tyr-Asn-Thr-

Tyr-Arg-Ser-Arg-Lys-Tyr-Xaa-Xaa-Xaa-Xaa-Val-Ala-Leu-Lys- Arg-Thr-Gly-Gln-Tyr-Lys-Leu-Gly-Xaa-Lys-Thr-Gly-Pro-Gly- Gln-Lys-Ala-Ile-Leu-Phe-Leu-Pro-Met-Ser-Ala-Lys-Ser, wherein Xaa in the 20-position is Ser, Thr or D-Ser, Xaa in the 21-position is Ser, Ala or D-Ser, Xaa in the 22- position is Trp or Met, Xaa in the 23-position is Tyr or Phe, Xaa in the 36-position is Pro or Ser, and one or more of the residues in positions 14 through 19 can be substituted by its D-isomer; provided however that the N- terminus can be shortened by deleting from 1 to 13 residues in sequence, that the C-terminus can be shortened by deleting from 1 to 30 residues in sequence, that an extension of up to 91 residues in the form appearing in a native mammalian bFGF peptide can be added at the N-terminus, and that the C-terminus can be optionally amidated, for use to prevent the uptake and infectivity of HSV in mammals.